In the cyanobacterium Synechocystis PCC 6803 and in the microalga Chlamydomonas reinhardtii, transient hydrogen photo-production is observed when cells are exposed to light in anoxia. We measured changes in H-2, O-2, and CO2 concentrations using time-resolved mass spectrometry in wild-type and mutant strains of Chlamydomonas and Synechocystis. In both organisms, non-photochemical reduction of the plastoquinone pool is shown to contribute to the initial H-2 photo-production. This pathway, which does not produce O-2, exhibits a low rate in normal conditions. From the effect of the uncoupler FCCP, we conclude that PS II-independent H-2 production in Chlamydomonas is limited by the trans-thylakoidal proton gradient. In Synechocystis, from the study of a mutant deficient in the NDH-I complex (M55), we conclude that PS II-independent H-2 production is limited by recycling of NAD(P)H through the NDH-I complex. Based on these conclusions, we propose strategies for optimising H-2 photo-production in these organisms. (C) 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.